1. Field of the Invention
The present invention relates to the field of culture media and methods for producing the same, and more particularly to culture media including pectin as the gelling agent.
2. Description of the Prior Art
A considerable variety and number of culture media are disclosed in the prior art. A corresponding number of methods for producing such media are also known. In general, media used for the growth of living cells, tissues or organisms contain certain ingredients. These ingredients include water, nutrients (generally a carbon source, a nitrogen source, and smaller amounts of other essential elements), buffers, and often a gelling or solidifying agent.
The majority of the biological media present in the prior art utilize agar, gelatin or silica gel as solidifying agents, also referred to herein as gelling agents. Disadvantages are associated with each of these materials as solidifying agents. Agar is obtained from marine algae which must be harvested from naturally occurring populations. The supply of agar correspondingly fluctuates from year to year, while the demand for solidifying agent continues to grow. The price for agar has steadily increased as a result, and the present price is relatively high. Another problem associated with the use of agar is the need to dispense the agar into its container while quite warm, since the agar solution may solidify at about 40.degree.-45.degree. C. A temperature of 45.degree. C. is too high for some cells to withstand without adverse effects.
Gelatin is easily obtained at a relatively reasonable cost, but it is easily hydrolyzed by many micro-organisms, which causes the gel to become a liquid. This is undesirable except in those cases where the hydrolysis is being used as a diagnostic biochemical test. Further, gelatin is generally available as a nutrient source for the organisms in contact with it, and as a result may interfere with the testing of specific nutrient sources. Gelatin also has the undesirable property of liquifying at quite low temperatures, so that media incorporating it as a gelling agent cannot be incubated above 25.degree. C. with assurance that the medium will retain its solid consistency. Disadvantages associated with silica gel include the relatively high cost of silica gel, and the complicated procedure required to prepare a medium using silica gel.
Pectins are routinely used as the thickening or gelling agent in the production of jams and jellies. However, the process generally used involves high sugar concentrations and low pH, neither of which is suitable for general microbial or tissue culture work. In fact, the high sugar and low pH characteristics are useful factors in preventing the establishment of growing, contaminating organisms in the jelly products.
Referring specifically to the prior art, there is disclosed in U.S. Pat. No. 2,970,948, issued to Stevens on Feb. 7, 1961, a culture medium in which pectin may be used. According to the procedure of the Stevens patent, a citrus serum agar culture is prepared by the following steps:
(1) adding pectinesterase to remove pectin by precipitation from fruit juice; PA1 (2) concentrating the fruit juice serum; PA1 (3) mixing with the fruit juice serum certain dry ingredients; and PA1 (4) drying and resulting mixture under a vacuum.
The Stevens patent discloses that included in the dry ingredients must be a gelling agent, which may include agar, gelatin or water soluble salts of pectic acid or alginic acid. It is further indicated that the gelling agent, which may include the pectic acid salts, is to be one which is capable of setting to a gel at room temperature upon cooling an aqueous solution thereof. In contrast to the method and media of the Stevens patent, the present invention involves the combination of a specified type of pectin and cations in the culture media container for biological growth.
In U.S. Pat. No. 2,373,729, issued to Willaman on Apr. 17, 1945, there is disclosed a thickening agent comprising a dry, powdered mixture of pectin, pectase and a water-soluble salt of a polyvalent metal. It is disclosed that the thickening agent is useful in the preparation of jellies, puddings, syrups, catsup, as well as non-food materials. The thickening agent is used by combining the dry-powdered mixture with an aqueous solution under certain conditions of temperature and pH. A different method for making a pectic preparation is disclosed in U.S. Pat. No. 2,540,050, issued to Leo and Taylor on Jan. 30, 1951. A gelatinous pectin/aluminun hydroxide co-precipitate is obtained from a pectin extract of fruit or vegetable material. The co-precipitate is then treated with pectase to provide the pectic material indicated to be useful in preparing jellies. In U.S. Pat. No. 3,360,440, issued to Haab et al., there is disclosed a cold water reconstitutable microbiological medium utilizing a modified cellulose as the sole gelling agent. U.S. Pat. No. 3,935,067, issued to Thayer on Jan. 27, 1976, discloses a culture media comprising inorganic water-swellable support material or water-absorbing clay mineral as a substitute for agar as a growth support and culture media.
A slow-set pectin is disclosed in U.S. Pat. No. 3,835,111, issued to Ehrlich and Cox on Sept. 10, 1974. The pectin material is prepared by contacting pectin with an ammoniacal alcohol solution at low temperature. The resulting pectin has reduced sensitivity to alkaline earth metal ions, which is indicated in the Ehrlich patent as producing a pectin suitable for preparation of sugar jellies. The pectin of the Ehrlich patent has a degree of methylation of 60-70%. A low methoxyl pectin is disclosed in U.S. Pat. No. 3,622,559, issued to Wiles and Smit on Nov. 23, 1971.
In U.S. Pat. No. 3,197,384, issued to Goldman on July 27, 1965, there is disclosed a process for determining the microbial sensitivity to certain anti-microbial agents. In the method of the Goldman patent, the anti-microbial agent is impregnated at premarked areas on a filter pad or sheet of filter paper. The pad or paper is then wetted with an inoculated broth and the reaction of the bacteria in the broth to the anti-microbial agent is perceivable overtime. In contrast to the procedure of the Goldman patent, the present invention relates in one aspect to the impregnation of filter paper or a similar support material with a multivalent metal cation material which will produce gelling of the pectin media when combined therewith.